Investigating capillary electrophoresis‐mass spectrometry for the analysis of common post‐translational modifications

EarlyView Article

Published: Mar 8, 2018

Author: Klaus Faserl, Bettina Sarg, Peter Gruber, Herbert H. Lindner

Journal: ELECTROPHORESIS

Abstract

Capillary electrophoresis coupled to mass spectrometry is a very efficient analytical method for the analysis of post‐translational
modifications because of its high separation efficiency and high detection sensitivity. Here we applied CE‐MS using three
differently coated separation capillaries for in‐depth analysis of a set of 70 synthetic post‐translationally modified peptides
(including phosphorylation, acetylation, methylation, and nitration). We evaluated the results in terms of peptide detection
and separation characteristics and found that the use of a neutrally coated capillary resulted in highest overall signal intensity
of singly modified peptides. In contrast, the use of a bare‐fused silica capillary was superior in the identification of multi‐phosphorylated
peptides (12 out of 15 were identified). Fast separations of approximately 12 min could be achieved using a positively coated
capillary, however, at the cost of separation efficiency. A comparison to nanoLC‐MS revealed that multi‐phosphorylated peptides
interact with the RP material very poorly so that these peptides were either washed out or elute as very broad peaks from
the nano column which results in a reduced peptide identification rate (7 out of 15). Moreover, the methods applied were found
to be very well suited for the analysis of the acetylated, nitrated and methylated peptides. All 36 synthetic peptides, which
exhibit one of those modifications, could be identified regardless of the method applied. As a final step in this study and
as a proof of principle, the phosphoproteome enriched from PC‐12 pheochromocytoma cells was analyzed by CE‐MS resulting in
5686 identified and 4088 quantified phosphopeptides. We compared the characterized analytes to those identified by a nanoLC‐MS
proteomics study and found that less than one third of the phosphopeptides were identical, which demonstrates the benefit
by combining different approaches quite impressively.